1. Mechanical Force Effects on Cellular Function
BioE506 –
James Eddy

2. Alexander Bershadsky, Michael Kozlov, and Benjamin Geiger
Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize
Alexander Bershadsky, Michael Kozlov, and Benjamin Geiger

3. Review Article Outline
Background – mechanosensitive adhesions
Focal adhesions (refresher)
Focal complex-to-adhesion transitions
Non-focal adhesion mechanosensors
Mechanisms of force-dependent transition
Physical models

4. Mechanosensitive Adhesions
Cells sense not only ligands & adhesion molecules, but mechanical cues:
mechanical forces (stresses)
deformations (strains)
“Just-in-time” mechanosensing
external perturbations unpredictable in extent and timing
strength of adhesion should adjust itself dynamically to amount of stress/strain applied to site

5. Focal Adhesions FAs associated with actin filaments in the cell
Interact with extracellular matrix via integrins
Evolve from focal complexes (FXs)

6. Force-dependent FX-to-FA Transition
The evidence:
Inhibition of myosin-II-driven contractility leads to accumulation of FXs and disappearance of FAs
Application of external force to FAs stimulates growth in direction of force – even with suppressed myosin II
Size of FAs and forces applied to them usually proportional
On soft matrix (strong forces not generated), large FAs not formed

7. Non-FA Mechanosensing Adhesions
Integrin-mediated
fibrillar adhesions:
evolve from FAs in force-dependent manner
do not assemble when force is relaxed
podosomes
lifespan, not shape, depends on substrate flexibility
Cadherin-mediated
cell-cell adherens junctions (AJs)
Platelet endothelial cell adhesion molecule (PECAM)

8. Molecular Mechanisms
Uncertain which molecular interactions (protein-protein) are regulated by force
Possible signaling mechanisms:
Modulation of phosphorylation-dependent protein-protein interactions
Recruitment/translocation of adaptor proteins
Regulation of GTPase pathways

9. Physical Modeling
In absence of detailed signaling models, scientists have used physical models to formulate “ground rules” for mechanosensitivity
Three specific models described:
Stress sensing
Strain sensing
Thermodynamic
Varying assumptions in three models:
Presence of a molecular “switch”
Location of mechanosensors
Protein dynamics

10. Stress-sensing Model
Protein switch: dragging force causes conformational change of mechanosensitive protein
Possible mechanism for maturation of FXs to FAs via ECM elasticity

11. Strain-sensing Model
Protein switch: compression of mechanosenosensitive protein layer leads to growth (front) and disassembly (rear) of adhesion plaque
“Crawling” or “treadmilling” motion
Few relative protein changes

12. Thermodynamic Model
Elastic stress within plaque decreases chemical potential, enhancing self-assembly by addition of new plaque molecules
Internal “treadmilling-like” motion of proteins which can progress in different directions
Abundant relative protein changes

13. Review Article Summary
Cells can sense mechanical stimuli, not just ligands or other molecules
Focal adhesions are one key example of mechanosensitive cell adhesion
Much is still unknown about protein interaction mechanisms in mechanosensitivity
Three models (stress-sensing, strain-sensing, thermodynamic), postulate physical “rules” for mechanosensitivity

14. Forced Unfolding of Proteins Within Cells
Colin P. Johnson, Hsin-Yao Tang, Christine Carag, David w. Speicher, Dennis E. Discher

15. Research Article Outline
Introduction
Spectrin proteins (red blood cells)
Cysteine labeling technique
Shear vs. Static labeling results (spectrin)
Labeling dynamics
Differential labeling in mesenchemal stem cells

16. Force-induced Protein Unfolding
Reversible domain unfolding has been shown to occur in adhesion proteins in response to external forces
Direct cell-level evidence is lacking, and a new “shotgun” approach for cysteine residue labeling is presented

17. Red Blood Cell – Spectrin Proteins
Spectrin proteins have been proven central to red blood cell deformability under stresses of blood flow
α and β chains crosslink with F-actin, and helical bundle domains unfold at low forces

18. Cysteine Labeling
Cysteine (Cys) residues are moderately hydrophobic, and often hidden in tertiary or quaternary protein structure
Protein unfolding allows labeling of newly exposed Cys residues by Cys-reactive phlorophore IAEDANS
Label phlorophores interact with exposed SH groups

19. Cysteine Labeling Shotgun in situ labeling:
Cells reversibly lysed, then resealed after entrapment of phlorophore IAEDANS
Dye-loaded cells either held static (with varying temperature), or sheared over physiological range of stresses
Cells relysed and imaged
Cells denatured, non-labeled Cys were alkylated with iodoacetamide (IAM)
Membranes separated by 1D SDS-PAGE

20. Shear vs. Static Cys Labeling
α and β chains show significant increase in Cys residue labeling when exposed to shear stresses
Other membrane proteins remain relatively unchanged

21. Shear vs. Static Cys Labeling
Liquid chromatography-coupled tandem mass spectrometry (LC MS/MS) was used to identify and quanitfy Cys-modified sites in spectrin bands (after excision, trypsinization)

22. Heat-induced Unfolding
Recombinant spectrin proteins were analyzed under varying temperatures

23. Labeling Dynamics
Sequential 2-dye labeling improves differential labeling

24. Mesenchemal Stem Cells
MSCs are contractile and strain underlying matrix in differentiation (stresses ~1000 times higher than fluid on RBCs)
Inhibition of non-muscle myosin II (NMM II) with the drug blebbistatin relaxes and softens cells, prevents differentiation

25. MSC Cys Labeling

26. MSC Cys Labeling
Much higher Cys labeling in tensed vs. relaxed NMM II (analogous to shear vs. static)
Blebbistatin induced depolymerization of vimentin and actin increases Cys labeling

27. Differentially Labeled NMMII Cys Sites
Homology model shows Cys90, identified as differentially labeled, to be buried between head and rod domains

28. Cys-labeling of Monomers/Polymers
It was shown that Cys labeling is enhanced for monomeric proteins (which agrees with depolymerization results)

29. Potential Cys-labeling Applications
Time-integrated Cys labeling techniques might be combined with other real-time imaging methods (e.g. FRET)
Could be used to correlate unfolding with post-translational modifications (i.e. phosphorylation)

30. Summary & Criticisms Certain membrains do unfold under physical forces
Cys labeling is an effective method for identifying and quantifying newly exposed protein regions following unfolding